Effects Of Four Novel Proteins Replacing Fishmeal On The Intestinal Health Of Largemouth Bass (Micropterus Salmoides)

Important for digestion and absorption in aquatic animals,the intestine breaks down macromolecular nutrients into small molecules that can be absorbed by the intestinal epithelium to promote growth and metabolism.It is particularly essential for aquatic animals to maintain a healthy intestine,as it is the greatest immune organ they possess.Both freshwater and marine aquaculture require significant quantities of fishmeal,but fishmeal is an extremely limited fishery resource,so finding alternatives to fishmeal is one of the aquaculture’s sustainable development objectives.This study was carried out on largemouth bass(Micropterus salmoides),a carnivorous fish with a protein requirement of over 40% and a commercial feed intake of 350g/kg-500g/kg fishmeal.Therefore,this study was conducted to investigate the nutritional and physiological responses and adaptation strategies of the intestinal tract of largemouth bass to novel proteins.We detected the effects of the novel proteins on the growth performance,serum biochemical parameters,intestinal histopathological changes,intestinal antioxidant capacity,intestinal inflammatory response,intestinal tight junction genes expression,intestinal apoptotic indexes expression,intestinal flora changes and intestinal short-chain fatty acids(SCFAs)content of largemouth bass by combining microbiome and metabolome techniques.This study gave a theoretical and practical foundation for the high-quality development of the farming industry by showing that novel proteins could be used to regulate the health of largemouth bass.1.The effects of complete replacement of fishmeal with four novel proteins on the intestinal health of large-size largemouth bassThe novel proteins i.e,Clostridium autoethanogenum(CAP),Tenebrio molitor(TM),Chlorella vulgaris(Ch M),and cottonseed protein concentrate(CSM),are promising food for the future aquaculture due to the limited fishmeal(FM)resources.However,in-depth investigations on the intestinal barriers of fish when fed novel proteins are sparse,and the association between intestinal flora and physiological biomarkers is still unknown.Five isonitrogenous and isolipidic diets comprising various proteins were developed,with fishmeal serving as the control and novel proteins as the experimental diets.Largemouth bass with an initial body weight of211.9±1.2g were used as experimental model and fed with these five diets for eight weeks.The findings from the current study indicated that the intestinal histological structure as well as immunochemistry barrier performance of the Ch M,CAP,and TM groups were superior to those of the CSM and FM groups,despite the fact that the villi height of FM was the greatest of all groups.Similarly,the intestinal inflammatory response and the permeability were elevated in CSM while decreased in Ch M,CAP,and TM group.Concerning the intestinal short fatty acids(SCFAs),the levels in the CAP as well as Ch M group were greater than the other proteins.From the data analysis of 16 S r RNA gene sequencing,the abundance of opportunistic pathogens including Ralstonia,Acinetobacter,Aeromonas,and Protebacteria were higher in CSM group while attenuated in Ch M,CAP and TM group.In addition,the abundance of nine types of intestinal-barriers-repairing probiotics were elevated in all novel proteins compared with the fishmeal group.These altered microbiota were highly correlated with intestinal inflammatory response,antioxidant status,immunochemistry barrier,physical barrier and SCFAs indexes by Mantel test and Pearson’s correlation analysis.Collectively,the ranking of the beneficial functions among these five proteins was Ch M > CAP > TM > FM > CSM according to Z-score and this study provides in-depth data manifesting the effects of novel proteins on the gut health status of large-size largemouth bass.2.The effects of alternate feeding between fishmeal and four novel proteins on the intestinal health of juvenile largemouth bassIn order to further optimize the above trial scenario,this section contained a further evaluation of the possibility of replacing fish meal with C.autoethanogenum,T.molitor,C.vulgaris and cottonseed protein concentrate from an alternate feeding perspective.Five isonitrogenous and isolipidic diets containing various proteins were manufactured,with a diet consisting of whole fishmeal serving as the control and diets containing novel proteins serving as the experimental diets.Largemouth bass with an initial body weight of 4.73 ±0.04 g employed as an experimental fish and given these five diets for the first 29 days followed by a fishmeal diet for the next 29 days.The diet of fishmeal,C.vulgaris,cottonseed protein concentrate,C.autoethanogenum,and T.molitor,was referred as FM/FM,Ch M/FM,CSM/FM,CAP/FM,and TM/FM,respectively.The results of current study showed that in the second stage of the study,all groups comprising the novel proteins outperformed the FM/FM group and had a higher specific growth rate(SGR)than in the first stage,but their overall growth performance was inferior to that of the FM/FM group.In terms of changes in intestinal pathology,the area of positive Alcian blue(AB)staining was decreased in the CSM/FM and CAP/FM groups after re-feeding of fish meal in the second stage compared to the first stage;additionally,the area of positive intestinal apoptosis was increased in the TM/FM group in the second stage,while it was decreased in the CAP/FM group.The Ch M/FM group increased intestinal antioxidant capacity on day 58,whereas the CSM/FM group decreased both intestinal and serum antioxidant capacity.Concerning apoptotic gene expression,the CAP/FM increased the anti-apoptotic gene bcl-2 transcript level under the alternate feeding strategy,whereas the other novel proteins did not respond significantly to the apoptotic gene expression.For intestinal permeability,the CAP/FM group reduced intestinal/serum IL-1β,TNF-α,and lipopolysaccharide levels,and elevated transcript levels of the tight junction protein claudin-4,thereby reducing intestinal permeability.In terms of intestinal immunocompetence,Ch M/FM promoted the secretion of intestinal lysozyme(LYS)and intestinal secretory immunoglobulin-like(s Ig A-like).For the microbiome and metabolome,the abundance of short-chain fatty acids,anaerobic bacteria,Lactococcus,and Firmicutes were significantly greater in the CAP/FM group than in the FM/FM group,whereas the abundance of aerobic bacteria,Pseudomonas,Streptococcus,and Proteobacteria was the lowest.However,no significant differences in intestinal flora or short-chain fatty acids were observed between the FM/FM group and the other groups fed novel proteins.In terms of microbial function prediction,CAP/FM could down-regulate lipid metabolic pathways and amino acid metabolic pathways in the intestine of largemouth bass,while promoting carbohydrate metabolic pathways.In addition,the microbiota were strongly connected with intestinal SCFAs,lipase activity,and tight junctions,as shown by the Mantel test and Pearson’s correlation.Taken together,according to Z-score,the ranking of advantageous functions among these protein diets was C.autoethanogenum diet > C.vulgaris diet >whole fishmeal diet > cottonseed protein concentrate > T.molitor diet.This study provides comprehensive data illustrating a mixed blessing effect of novel protein diets on the gut health of juvenile largemouth bass under an alternate feeding strategy.